Superoxide anion and nitric oxide in high-grade esophagitis induced by acid and pepsin in rabbits.

It has been proposed that free radicals are involved in the pathogenesis of esophageal mucosal damage induced by acid and pepsin. Recent data have suggested that nitric oxide (NO) is involved in the mucosal defense of the esophagus and that superoxide anion plays a minor role in low-grade esophagitis. To study the role and potential interaction of NO and superoxide anion in an experimental model of high-grade esophagitis, acidified pepsin was perfused (45 min/12 hr) for five days in rabbits with different agents to modulate the generation of these radicals. Measurements included both macroscopic and microscopic mucosal damage, superoxide anion generation, NO synthase mucosal activity, and peroxynitrite formation. High-grade esophagitis was associated with mucosal superoxide anion generation. Treatment with exogenous superoxide dismutase completely prevented mucosal damage. The perfusion of acidified pepsin in the lumen of the esophagus was initially associated with increased NO synthase mucosal activity but decreased with the progression of damage. Generation of peroxynitrites was present in those cases with severe damage. Treatment with NO-modifying agents did not induce consistent modification of mucosal damage. It is concluded that superoxide anion is involved in the induction of high-grade esophagitis and that it interacts with nitric oxide to generate peroxynitrite radicals in this model. Superoxide dismutase but not NO-donor-modifying agents might have a therapeutic role in preventing severe esophageal mucosal damage induced by acid and pepsin.

Nitric oxide and superoxide anion in low-grade esophagitis induced by acid and pepsin in rabbits.

It has been suggested that free radicals are involved in esophagitis. To study the role and potential interaction of superoxide anion and nitric oxide (NO) in low-grade esophagitis, we perfused acidified pepsin (30 min every 12 hr) for seven days in rabbits treated with different agents to modulate the generation of these radicals. Measurements included macroscopic and microscopic damage, superoxide anion generation, mucosal nitric oxide synthase activity, and peroxynitrite formation. Low-grade esophagitis was associated with increased nitric oxide synthase mucosal activity and mucosal damage was dose-dependently increased by treatment with the NO synthase inhibitor NG-nitro-L-arginine. Superoxide anion was scarcely generated in the mucosa, but this was not accompanied by any change in the activity of mucosal superoxide dismutase. Treatment with superoxide dismutase did not improve mucosal damage. Generation of peroxynitrites was not detected. In conclusion, nitric oxide is involved in the mucosal defense of the esophagus against acid- and pepsin-induced damage. Superoxide anion generation seems irrelevant in the induction of low-grade esophagitis and not sufficient to interact with nitric oxide to generate measurable mucosal peroxynitrite radicals.

Omeprazole does not interfere with the antiplatelet effect of low-dose aspirin in man.

BACKGROUND: Experimental studies have shown that omeprazole and other anti-inflammatory agents compromise the therapeutic activity of nonsteroidal anti-inflammatory drugs and aspirin in rats. It is not known whether this effect will occur in humans. Our aim was to determine whether omeprazole affects the antiplatelet effects of low-dose aspirin in humans. METHODS: Platelet lumiaggregation, skin bleeding time, plasma levels of aspirin and salicylic acid, and serum gastrin levels were determined in 14 healthy men before and after the ingestion of 125 mg aspirin with or without previous treatment with 20 mg/day of omeprazole for 4 days before testing. RESULTS: Omeprazole increased serum gastrin levels from 64 (median; range, 52-91) pg/ml to 80.5 (median; range, 56-455) pg/ml (P < 0.05) but did not significantly affect the plasma concentration of aspirin or salicylic acid. Aspirin increased skin bleeding time in all subjects, and the increase was similar with and without previous omeprazole treatment. Aspirin inhibited platelet aggregation in response to both collagen and arachidonic acid regardless of omeprazole treatment. CONCLUSION: A single dose of 20 mg omeprazole daily does not interfere with the biologic activity of 125 mg aspirin on platelets in humans.

Neutrophil dynamics in abdominal cavity of peritonitic rats treated with antiseptics.

Peritonitis was induced in Wistar rats by intraperitoneal inoculation of pure Escherichia coli. Rats in which 2 ml of 1% povidone iodine had been injected intraperitoneally 5 min after the bacterial challenge, showed a decrease in the neutrophil percentage of the peritoneal cell population during the first hours of peritonitis compared to the control group. When the same experiment was performed with 2 ml of 0.05% chlorhexidine the percentage of neutrophils was superior to the control group in the first hours after bacterial challenge. These results concur with a previous study in which the deleterious effect of povidone iodine and the beneficial effect of chlorhexidine were demonstrated.

Development of hepatic microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase in the young chick.

1. The 3-hydroxy-3-methylglutaryl-CoA reductase activity increased from 1 to 4 weeks of age, but decreased from 4 to 8 weeks of age. 2. Cholesterol 7 alpha-hydroxylase activity increased from 1 to 4 weeks, decreased from 4 to 6 weeks, and increased again from 6 to 8 weeks of age. 3. Serum total and free cholesterol concentrations decreased from 1 to 6 weeks of age, but increased from 6 to 8 weeks of age.

Changes in breast muscle composition in the young chick.

Nitrogen, protein and nucleic acids concentrations in the breast muscle (pectoralis thoracica), composition of sarcoplasmic and myofibrillar protein, and serum level of 3-methylhistidine were determined in female Warren chicks at 2, 3, 4 and 6 wk of age. Sarcoplasmic nitrogen and protein concentrations showed opposite changes to those presented by myofibrillar concentrations, with a maximum value at 3 and 4 wk of age and decreasing at 6 wk of age. Ribonucleic acid concentration decreased and protein/ribonucleic acid ratio increased from 2 to 6 wk of age. Differences were observed in the electrophoretical banding pattern of myofibrillar protein at 3 wk of age. Serum level of 3-methylhistidine increased from 2 to 4 wk of age (from .71 to 3.25 nmol/g), and no change was observed at 6 wk of age (3.46 nmol/g).

Changes in the concentration and composition of biliary and serum bile acids in the young domestic fowl.

1. Concentrations of biliary and serum bile acids, their molecular compositions and serum cholesterol concentrations were determined in chicks at 2, 3, 4 and 6 weeks of age. 2. The concentration of biliary bile acid was maximal at 3 to 4 weeks, decreasing by 6 weeks of age. 3. The serum concentration of bile acid was maximal at three weeks of age. 4. Serum total cholesterol increased from two weeks and was maximal at 6 weeks of age. 5. Chenodeoxycholic acid was the predominant biliary unconjugated bile acid. 6. Tauro-chenodeoxycholic acid and tauro-cholic acid were the dominant molecular species of biliary and serum conjugated bile acid.

Increased levels of platelet-activating factor in blood from patients with cirrhosis of the liver.

The levels of platelet-activating factor (paf-acether) were measured in blood and ascitic fluid from cirrhotic patients and in blood from a group of controls, using a recently described technique for extraction and measurement. In addition, activity of acetylhydrolase, the main catabolic enzyme for paf-acether, was also measured. The highest levels of paf-acether in blood were found in decompensated cirrhotics (1.78 +/- 0.62 ng ml-1; mean +/- SD, n = 8). Compensated cirrhotics showed lower blood values (0.79 +/- 0.21, n = 4), but higher than controls (0.20 +/- 0.04, n = 12). Paf-acether levels in ascitic fluid were similar to those of blood. Values of acetylhydrolase in serum were similar in all the groups studied (3.0 +/- 0.4 in cirrhotics vs. 2.3 +/- 0.4 nmol min-1 mg-1 of protein in controls). These data suggest an enhanced production of paf-acether in cirrhotic patients rather than a decreased catabolism. High levels of paf-acether in blood could be involved in the impaired haemodynamics of cirrhotic patients and in their renal function alterations.

Evidence of a role for PAF-acether in the pathophysiology of the shock state.

The pathophysiology of the shock state includes a variety of hemodynamic changes such as systemic hypotension, pulmonary hypertension and increased vascular permeability leading to the extravasation of protein rich plasma. These changes can be initiated by different etiological factors, but many of them have been related to the stimulation of activation systems (complement, kinins, etc.) or to the generation of inflammatory mediators. The purpose of the present study has been to obtain evidence of the involvement of paf-acether in the pathogenesis of the shock state initiated in rat and mouse by Gram-negative bacteria and soluble aggregates of immunoglobulin G. The injection of 1-2 MDa aggregates of immunoglobulin G to normal Sprague-Dawley rats, induced a dose-dependent systemic hypotension which appeared about five minutes after completion of the intravenous challenge. Simultaneously, extravasation of protein-rich plasma occurred as judged from the finding of an increased clearance of 125I-BSA. In similar experiments in mice, a reduction of the vascular volume was observed using 51Cr-labelled homologous red blood cells. Under these conditions, a lipid compound analogous to paf-acether was obtained from the liver and the spleen of these animals. The generation of this compound preceded the development of blood volume depletion and could be suppressed by either quinacrine or depletion of mononuclear phagocytes by total irradiation with 700 rads. The previous treatment of the rats with the compound BN 52021 (a specific antagonist of the paf-acether receptor) at a dose of 5mg/kg, i.v., prevented the appearance of hypotension and extravasation in response to an i.v. challenge with soluble aggregates of immunoglobulin G. Interestingly, the reversal of hypotension was also observed when BN 52021 was infused after the immunoaggregates (5mg/kg). The possible involvement of paf-acether in the hemodynamic changes of Gram-negative sepsis was studied in rats which had received an intraperitoneal inoculation of E. coli. The animals inoculated with the doses of bacteria which produced mortality showed a time- and dose-dependent increase of vascular permeability as judged from the presence of abundant peritoneal exudate and the reduction of the circulating volume. Simultaneously, significant amounts of paf-acether could be obtained from the peritoneal exudate and from the spleen preceding to the development of the circulating volume depletion.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of PAF-acether and blood volume changes in gram-negative sepsis.

Gram-negative sepsis was induced in rats by intraperitoneal injection of Escherichia coli. The development of bacterial peritonitis and septicemia was monitored by counting the number of peritoneal cells and by performing cultures of blood samples. Mortality reached a 50% rate when rats were injected with 2 X 10(8) colony-forming units. Rats injected with the doses of bacteria which induced mortality showed a time- and dose-dependent increase of vascular permeability as judged by the presence of abundant peritoneal exudate and by the depletion of the circulating volume. In order to know whether the generation of PAF-acether could be involved in the development of the permeability changes, the formation of this mediator was measured in the peritoneal cells and spleen of animals at different times and in response to different doses of E. coli. Significant amounts of PAF-acether could be obtained preceding the development of blood volume depletion in response to the injection of doses of E. coli which induced both mortality and the development of permeability. These data suggest that PAF-acether might be one of the inflammatory mediators involved in the pathogenesis of the hemodynamic changes observed in endotoxemia.

Characteristics of the binding of platelet-activating factor to platelets of different animal species.

The binding of platelet-activating factor (PAF-acether) to human, rabbit and rat platelets was investigated by incubating washed platelets at 37 degrees C with radiolabeled [3H]PAF-acether. Scatchard plot analysis of the binding showed that human and rabbit platelets possess two different types of binding sites. One of them was saturable and of high affinity (KD 1.58 +/- 0.36 nM in human and 0.9 +/- 0.5 nM in rabbits), and the other one had nearly infinite binding capacity. By contrast, rat platelets only showed non-specific binding. Addition of unlabeled PAF-acether 5 min after the addition of [3H]PAF-acether showed that internalization of 66 +/- 9% of the specific binding in human and 52 +/- 6.9% in rabbit platelets had occurred. Specific binding of [3H]PAF-acether in rabbit platelets correlated well with [3H]serotonin release in response to different doses of PAF-acether and with the uptake of calcium by the platelets. By contrast, PAF-acether did not induce [3H]serotonin release or calcium uptake by rat platelets. The following data suggest that the potential of PAF-acether to activate platelets depends on the interaction with a specific membrane receptor rather than on a non-receptor-mediated alteration of the platelet membrane: (1) platelets from animal species sensitive to PAF-acether show saturability and specificity of binding; (2) platelets from one animal species non-sensitive to PAF-acether lack specific binding; (3) PAF-acether does not induce calcium uptake by platelets from an animal species which lacks specific binding sites.

Presence of platelet-activating factor in blood from humans and experimental animals. Its absence in anephric individuals.

Blood from humans and experimental animals was examined for the presence of platelet-activating factor. The procedure included a rapid extraction of the samples and a purification of the lipid content by thin layer chromatography. The chemical characterization was performed by phospholipases' treatment and high performance liquid chromatography. Rats contained the highest concentrations of platelet-activating factor and rabbits the lowest. Five anephric patients undergoing support hemodialysis had undetectable blood levels and the same finding was observed in six rats in which surgical nephrectomy was performed. Based on these data we suggest: 1) Low amounts of platelet-activating factor can be present in blood under normal conditions. 2) Kidney tissue seems to play a role in the formation of the platelet-activating factor that can be detected in blood under these conditions.

The role of calcium ions in the process of acetyltransferase activation during the formation of platelet-activating factor (PAF-acether).

The role of Ca2+ in the activation of the enzyme lyso-(platelet-activating factor): acetyl-CoA acetyltransferase was studied in rat peritoneal macrophages in response to complement-coated zymosan particles and ionophore A23187. By using Ca2+-containing buffers, a threshold concentration of extracellular Ca2+ above 1 microM was found to be necessary to observe the activation of the enzyme in response to zymosan. By contrast, a significant role of intracellular Ca2+ in this process could be ruled out, since the putative intracellular calcium-transport antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] did not inhibit the activation of the acetyltransferase induced by zymosan in the presence of extracellular Ca+. The link between acetyltransferase activation and extracellular Ca2+ transport was studied by measuring Ca2+ uptake in response to the stimuli. Zymosan particles induced a rapid increment in cell-associated Ca2+ which correlated well with the extent of acetyltransferase activation (r = 0.91) and with the release of platelet-activating factor (r = 0.95) in response to different doses of zymosan. Cellular Ca2+ efflux in response to zymosan particles was also measured and found to be increased, as compared with controls, when the activation of the acetyltransferase declined. In short, the data suggest that the entry of extracellular Ca2+ into the cell is a crucial event in the activation of acetyltransferase and, thereby, in the formation of platelet-activating factor in rat peritoneal macrophages.

Platelet-activating factor: an effector substance of the vasopermeability changes induced by the infusion of immune aggregates in the mouse.

The effect of intravenous infusion of heat-aggregated IgG on vascular permeability was studied by using 51Cr-labelled homologous red blood cells in mice. Simultaneously, the recovery of platelet-activating factor (PAF) from the mononuclear phagocytic system (MPS) was attempted. Whereas PAF was found only in trace amounts in the liver of control animals, the infusion of aggregates induced the release of PAF from liver and spleen in a time- and dose-dependent manner. A possible link between PAF release and permeability is sustained on the following basis. PAF release precedes permeability changes and both show a parallel dose-response pattern plateauing for doses higher than 1 mg. Further, depletion of mononuclear phagocytes by total irradiation with 700 rads, or pharmacological blockade of phospholipases by prior treatment with quinacrine, induced abrogation of permeability changes and PAF release from spleens, together with an 80% reduction of the amount of PAF obtained from livers. These data suggest the following conclusions: 1) PAF release may occur in vivo when the MPS is stimulated by phagocytosable material; 2) PAF seems to be an effector substance of the permeability changes which occur during the administration of immune aggregates.

Presence in normal human urine of a hypotensive and platelet-activating phospholipid.

Urine from normotensive volunteers and patients with systemic lupus erythematosus glomerulonephropathy was sequentially concentrated by negative-pressure ultrafiltration, dialyzed against distilled water, and extracted into the chloroform phase of a mixture of organic solvents(chloroform:methanol:water, 1:1:0.9 vol/vol). The lipid fraction was further purified by thin-layer chromatography on silica gel plates using neutral, acidic, and basic mixtures of organic solvents and it was then tested for its ability to induce the release of [3H]serotonin from rabbit platelets. All of the samples contained a platelet-activating moiety similar to a synthetic platelet-activating factor (PAF-acether) on the basis of its chromatographic behavior, resistance to the pretreatment of platelets by 10(-6) M indomethacin, and loss of activity by alkaline methanolysis or treatment by phospholipases A2, C, and D. Cross-densensitization experiments between synthetic PAF-acether and the urine factor showed that both compounds act on platelets through the activation of the same putative receptor. Further, the urine factor induced hypotension when intra-arterially injected in normotensive rats, and this activity was also abrogated by alkaline methanolysis. In summary, these data provide evidence of the presence in normal human urine and, probably, of the release by the kidney of a lipid factor with platelet-activating and hypotensive activity whose general structure seems to be alkyl-acyl-glyceryl-phosphorylcholine and, therefore, is similar to the structure of the inflammatory mediator PAF-acether and the antihypertensive polar renomedullary lipid.

Vascular actions of synthetic PAF-acether (a synthetic platelet-activating factor) in the rat: evidence for a platelet independent mechanism.

Since rat platelets fully responsive to thrombin and collagen did not respond by releasing 3H-serotonin with up to 10 micrograms/ml of synthetic PAF-acether, the rat, contrariwise to the rabbit, was considered to be an appropriate model to study the actions of PAF-acether not mediated through the activation of platelets and the subsequent release of their inflammatory mediators. We developed an experimental approach using 57Co and 113Sn radiolabeled microspheres to assess the effect of PAF-acether on cardiac output, peripheral vascular resistance, and regional flows and resistance. The effect on vascular permeability and blood volume was studied by measuring the clearance of 125I-HSA and the variations of the hematocrit. A significant fall in blood pressure and peripheral vascular resistance was found with doses of PAF-acether ranging from 0.05 to 5 micrograms. Moreover, the higher doses of PAF-acether also induced a marked depletion of blood volume. A significant fall in spleen, coronary, and kidney output, but not in cardiac output, was also found. Our data show that PAF-acether, by itself, induces a drop in peripheral vascular resistance and, at higher doses, also in circulating volume, accounting for both by the hypotensive effect. The redistribution of cardiac output seems to be the expression of a nonuniform action of the compound on the vascular resistance of the different organs.

Non-platelet-mediated vascular actions of 1-O-alkyl-2-acetyl-sn-3-glyceryl phosphorycholine (a synthetic PAF).

Rat platelets fully responsive to thrombin and collagen did not release 3H-serotonin with up to 10 microgram/ml synthetic PAF. Therefore, an experimental approach using 57Co and 113Sn radiolabelled microspheres was developed to evaluate the effect of PAF on cardiac output (CO), peripheral vascular resistance (PVR) and redistribution of CO among organs. The effect on vascular permeability was studied by measuring the clearance of 125I-HSA and the variations of the haematocrit. A significant fall in blood pressure and PVR was found with doses of PAF from 50 to 5000 ng. Moreover, the highest doses of PAF induced also a marked reduction in blood volume. A significant fall in spleen, coronary and kidney output was found but not in CO. Our data show that PAF, by itself, induces a fall in PVR and at higher doses also in circulating volume, both accounting for the hypotensive effect. The redistribution of CO seems to be the expression of a non-uniform action upon PVR.